A number of genes have been implicated in the process of malignant transformation in the mammary glands of experimental animals and humans. The mouse mammary gland provides an excellent experimental model within which to elucidate the function of these putative mammary oncogenes during the growth, development, and differentiation of mammary tissue in vivo. Any portion of the adult mouse mammary gland at any age and throughout all stages of differentiation can repopulate the gland-free mammary fat pad of syngeneic mice upon transplantation. Similar results are obtained when dissociated glandular epithelium is injected into the fat pad or alternatively grown in culture under prescribed conditions prior to reintroduction into the mammary fat pad. We have initiated experiments designed to evaluate the feasibility of introducing functional genetic elements into mouse mammary cells with recombinant retroviral expression vectors antecedent to the reintroduction of these cells into a gland-free fat pad. Paramount to the success of such a project is the maintenance of the "repopulation potential" of the mammary epithelial cells. Our studies and those of others indicate that the number of cells capable of repopulation of the fat pad is small initially and the ability to repopulate is gradually lost through mitotic events. We have morphologically identified a potential candidate for these "mammagenic" cells in situ and in explant cultures. These cells seem to differentially express certain cytokeratins during growth and expansion of the mammary gland in vivo, suggesting that specific keratin expression could serve as a marker for these "stem" cells. In addition, primary mammary cell cultures are being evaluated for the efficiency of retroviral gene transfer, the level of recombinant trans-gene expression in the infected cultures, and the stability of recombinant retroviral gene expression in "mammagenic cell populations" subsequent to repopulation of the gland-free mammary fat pads. We have developed a number of partially transformed preneoplastic mammary outgrowth lines which are carried serially in vivo by transplantation into gland-free mammary fat pads. Cells from these lines have been grown in vitro and are capable of repopulating mammary fat pads subsequently. These cells are being tested for retroviral gene transfer studies.